U.S. patent application number 15/565987 was filed with the patent office on 2018-04-19 for crosslinked polymer structure and use for same.
The applicant listed for this patent is NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY. Invention is credited to Toshiyuki KANAMORI, Kana MORISHITA, Kimio SUMARU, Toshiyuki TAKAGI.
Application Number | 20180104940 15/565987 |
Document ID | / |
Family ID | 57126277 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180104940 |
Kind Code |
A1 |
SUMARU; Kimio ; et
al. |
April 19, 2018 |
CROSSLINKED POLYMER STRUCTURE AND USE FOR SAME
Abstract
A method for producing a structure composed of crosslinked
polymer, including laminating a photo-dissoluble polymer layer on a
substrate; laminating a layer containing a crosslinkable polymer on
the photo-dissoluble polymer layer; irradiating the layer
containing the crosslinkable polymer with light in a patternwise
manner under crosslinking conditions to crosslink the crosslinkable
polymer in a patternwise manner to obtain a crosslinked polymer
sheet; washing and removing a uncrosslinked crosslinkable polymer
to obtain a patterned crosslinked polymer sheet; and irradiating
the photo-dissoluble polymer layer with light in a patternwise
manner under dissolution conditions to dissolve the
photo-dissoluble polymer layer in a patternwise manner, and peeling
the crosslinked polymer sheet in a patternwise manner from the
substrate.
Inventors: |
SUMARU; Kimio; (Tsukuba-shi,
JP) ; TAKAGI; Toshiyuki; (Tsukuba-shi, JP) ;
MORISHITA; Kana; (Tsukuba-shi, JP) ; KANAMORI;
Toshiyuki; (Tsukuba-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND
TECHNOLOGY |
Tokyo |
|
JP |
|
|
Family ID: |
57126277 |
Appl. No.: |
15/565987 |
Filed: |
April 15, 2016 |
PCT Filed: |
April 15, 2016 |
PCT NO: |
PCT/JP2016/062099 |
371 Date: |
October 12, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B32B 27/16 20130101;
B32B 2255/10 20130101; C12M 25/02 20130101; G03F 7/038 20130101;
G03F 7/325 20130101; G03F 7/039 20130101; C08L 101/06 20130101;
G03F 7/2024 20130101; G03F 7/095 20130101; B32B 27/00 20130101;
C12M 25/14 20130101; C12M 3/00 20130101; G03F 7/32 20130101 |
International
Class: |
B32B 27/16 20060101
B32B027/16; C08L 101/06 20060101 C08L101/06; G03F 7/095 20060101
G03F007/095 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2015 |
JP |
2015-085322 |
Claims
1. A method for producing a structure composed of crosslinked
polymer, comprising: laminating a photo-dissoluble polymer layer on
a substrate; laminating a layer containing a crosslinkable polymer
on the photo-dissoluble polymer layer; irradiating the layer
containing the crosslinkable polymer with light in a first
patternwise manner under crosslinking conditions to crosslink the
crosslinkable polymer in the first patternwise manner to obtain a
crosslinked polymer sheet; washing and removing a uncrosslinked
crosslinkable polymer to obtain a crosslinked polymer sheet
patterned in the first patternwise manner; and irradiating the
photo-dissoluble polymer layer with light in a second patternwise
manner under dissolution conditions to dissolve the
photo-dissoluble polymer layer in the second patternwise manner,
and peeling the crosslinked polymer sheet from the substrate.
2. The method for producing the structure composed of crosslinked
polymer according to claim 1, further comprising: breaking and
removing the crosslinked polymer sheet peeled from the
substrate.
3. The method for producing the structure composed of crosslinked
polymer according to claim 1, wherein the crosslinkable polymer is
a compound having a plurality of hydroxyl groups and having a
weight-average molecular weight of 2,000 or more.
4. The method for producing the structure composed of crosslinked
polymer according to claim 1, wherein the crosslinkable polymer is
water-soluble.
5. The method for producing the structure composed of crosslinked
polymer according to claim 1, wherein the crosslinkable polymer is
a polysaccharide or a derivative thereof.
6. (canceled)
7. A structure composed of crosslinked polymer, comprising: a
substrate; a photo-dissoluble polymer layer laminated on the
substrate and patterned in a reversed pattern of a second
patternwise manner; and a crosslinked polymer sheet, at least one
portion thereof is laminated on the photo-dissoluble polymer layer,
wherein the crosslinked polymer sheet is patterned in a first
patternwise manner and peeled from the substrate in the second
patternwise manner.
8. The structure composed of crosslinked polymer according to claim
7, wherein a peeled portion of the crosslinked polymer sheet is
removed and the substrate is exposed at the peeled portion and the
exposed part of the substrate forms a cell adhesivable portion, and
the remaining crosslinked polymer sheet forms a non-cell
adhesivable portion.
9. The structure composed of crosslinked polymer according to claim
8, wherein the cell adhesivable portion and the non-cell
adhesivable portion are linearly arranged in parallel with each
other.
10. The structure composed of crosslinked polymer according to
claim 8, wherein the cell adhesivable portion has a plurality of
island-like portions and connecting portions for connecting
together the plurality of the island-like portions.
11. The structure composed of crosslinked polymer according to
claim 10, wherein all of the island-like portions have
substantially the same area.
12. A cell culturing method for uniformly culturing cells,
comprising: a step of culturing cells on the surface of the
structure composed of crosslinked polymer according to claim 11,
with the cells being adhered to the plurality of the island-like
portions.
13. A method for producing a cell mass, comprising: a step of
culturing cells on the surface of the structure composed of
crosslinked polymer according to claim 11; and a step of detaching
the cells to obtain substantially the same number of cell masses as
the number of the island-like portions consisting of the cells
being adhered to the plurality of the island-like portions.
14. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to a structure composed of
crosslinked polymer and use thereof. More specifically, the present
invention relates to a method for producing a structure composed of
crosslinked polymer, a laminate for producing a structure composed
of crosslinked polymer, a structure composed of crosslinked
polymer, a structure composed of crosslinked polymer for cell
culture, a cell culture method, a method for producing a cell mass,
and a cell mass. Priority is claimed on Japanese Patent Application
No. 2015-085322, filed on Apr. 17, 2015, the content of which is
incorporated herein by reference.
BACKGROUND ART
[0002] Applications of materials having a microstructure on the
surface thereof have been actively studied in various fields
including biotechnology and medical care. Particularly, in
microfluidic systems, devices, and the like handling cells or the
like, attention has been paid to a .mu.m-scale structure forming
technique. For example, Patent Document 1 discloses a micro-device
for a cellular tissue body, including a cell holding chamber for
holding cells, in which the cell holding chamber has a base
including at least one tissue body-forming region, an inlet port
for charging a culture fluid into the cell holding chamber and an
outlet port for discharging the culture fluid from the cell holding
chamber; and the tissue body-forming region includes one first
region exhibiting cell adhesiveness and a second region surrounding
the first region and exhibiting lower cell adhesiveness than that
of the first region, and has an area of 100 to 1.times.10.sup.6
.mu.m.sup.2.
CITATION LIST
Patent literature
[0003] [Patent Document 1] Japanese Patent No. 4033265
SUMMARY OF INVENTION
Technical Problem
[0004] With such a background, development of a microstruture
forming technique is expected. Therefore, it is an object of the
present invention to provide a novel microstructure forming
technique. More specifically, the object of the present invention
is to provide a method for producing a structure composed of
crosslinked polymer, a laminate for producing a structure composed
of crosslinked polymer, a structure composed of crosslinked
polymer, a structure composed of crosslinked polymer for cell
culture, a cell culture method, a method for producing a cell mass,
and a cell mass.
Solution to Problem
[0005] The present invention includes the following aspects.
[0006] (1) A method for producing a structure composed of
crosslinked polymer, including: a step (a) of laminating a
photo-dissoluble polymer layer on a substrate; a step (b) of
laminating a layer containing a crosslinkable polymer on the
photo-dissoluble polymer layer; a step (e) of irradiating the layer
containing the crosslinkable polymer with light in a patternwise
manner under crosslinking conditions to crosslink the crosslinkable
polymer in a patternwise manner to obtain a crosslinked polymer
sheet; a step (d) of washing and removing a uncrosslinked
crosslinkable polymer to obtain a patterned crosslinked polymer
sheet; and a step (e) of irradiating the photo-dissoluble polymer
layer with light in a patternwise manner under dissolution
conditions to dissolve the photo-dissoluble polymer layer in a
patternwise manner, and peeling the crosslinked polymer sheet in a
patternwise manner from the substrate.
[0007] (2) The method for producing the structure composed of
crosslinked polymer according to (1), further including a step (f)
of breaking and removing the crosslinked polymer sheet peeled in a
patternwise manner from the substrate.
[0008] (3) The method for producing the structure composed of
crosslinked polymer according to (1) or (2), in which the
crosslinkable polymer is a compound having a plurality of hydroxyl
groups and having a weight-average molecular weight of 2,000 or
more.
[0009] (4) The method for producing the structure composed of
crosslinked polymer according to any one of (1) to (3), in which
the crosslinkable polymer is water-soluble.
[0010] (5) The method for producing the structure composed of
crosslinked polymer according to any one of (1) to (4), in which
the crosslinkable polymer is a polysaccharide or a derivative
thereof.
[0011] (6) A laminate for producing a structure composed of
crosslinked polymer, including: a substrate; a photo-dissoluble
polymer layer laminated on the substrate; and a layer containing a
crosslinkable polymer laminated on the photo-dissoluble polymer
layer.
[0012] (7) A structure composed of crosslinked polymer, including:
a substrate; and a crosslinked polymer sheet provided on the
substrate, in which the substrate and the crosslinked polymer sheet
have an adhesive region where the substrate and the crosslinked
polymer sheet are adhered, and a non-adhesive region where the
substrate and the crosslinked polymer sheet are not adhered, in a
planar view, the adhesive region of the substrate is closed to
surround the periphery of the non-adhesive region of the substrate,
and the non-adhesive region of the crosslinked polymer sheet is
provided with at least one through-hole.
[0013] (8) A structure composed of crosslinked polymer for cell
culture, including: a substrate; and a cell adhesivable portion and
a non-cell adhesivable portion provided in a patternwise manner on
the substrate, in which the substrate is exposed in the ceil
adhesivable portion, and the non-cell adhesivable portion is made
of a crosslinked polymer sheet adhered on the substrate.
[0014] (9) The structure composed of crosslinked polymer for cell
culture according to (8), in which the cell adhesivable portion and
the non-cell adhesivable portion are linearly arranged in parallel
with each other.
[0015] (10) The structure composed of crosslinked polymer for cell
culture according to (8), in which the cell adhesivable portion has
a plurality of island-like portions and connecting portions for
connecting together the plurality of the island-like portions.
[0016] (11) The structure composed of crosslinked polymer for cell
culture according to (10), in which all of the island-like portions
have substantially the same area.
[0017] (12) A cell culturing method for uniformly culturing cells,
including: a step of culturing cells on the surface of the
structure composed of crosslinked polymer for cell culture
according to (11), with the cells being adhered to the plurality of
the island-like portions.
[0018] (13) A method for producing a cell mass, including: a step
of culturing cells on the surface of the structure composed of
crosslinked polymer for cell culture according to (11); and a step
of detaching the cells to obtain substantially the same number of
cell masses as the number of the island-like portions consisting of
the cells being adhered to the plurality of the island-like
portions.
[0019] (14) A cell mass obtained by the production method according
to (13).
Advantageous Effects of Invention
[0020] According to the present invention, it is possible to
provide a novel microstructure forming technique. More
specifically, the present invention is capable of providing a
method for producing a structure composed of crosslinked polymer, a
laminate for producing a structure composed of crosslinked polymer,
a structure composed of crosslinked polymer, a structure composed
of crosslinked polymer for cell culture, a cell culture method, a
method for producing a cell mass, and a cell mass.
BRIEF DESCRIPTION OF DRAWINGS
[0021] FIG. 1A is a confocal laser scanning micrograph showing a
structure composed of crosslinked polymer produced in Example
2.
[0022] FIG. 1B is a confocal laser scanning micrograph showing a
structure composed of crosslinked polymer produced in Example
2.
[0023] FIG. 1C is a confocal laser scanning micrograph showing a
structure composed of crosslinked polymer produced in Example
2.
[0024] FIG. 1D is a confocal laser scanning micrograph showing a
structure composed of crosslinked polymer produced in Example
2.
[0025] FIG. 1E is a confocal laser scanning micrograph showing a
structure composed of crosslinked polymer produced in Example
2.
[0026] FIG. 2 is an optical micrograph showing a structure composed
of crosslinked polymer produced in Example 2.
[0027] FIGS. 3(a) to 3(f) are diagrams showing a method for
producing a structure composed of crosslinked polymer.
[0028] FIG. 4 is an optical micrograph showing the results of
Example 3.
[0029] FIG. 5A is an optical micrograph showing the results of
Example 4.
[0030] FIG. 5B is an optical micrograph showing the results of
Example 4.
[0031] FIG. 6 is an optical micrograph of MDCK cells cultured in
Example 5.
[0032] FIG. 7A is an optical micrograph of NIH/3T3 cells cultured
in Example 5.
[0033] FIG. 7B is an optical micrograph of NIH/3T3 cells cultured
in Example 5.
[0034] FIG. 8 is an optical micrograph of human iPS cells cultured
in Example 5.
DESCRIPTION OF EMBODIMENTS
[0035] Hereinafter, embodiments of the present invention will be
described in detail with reference to the accompanying drawings as
needed. The dimensions in the drawings are partly exaggerated for
explanation purposes and do not always correspond to actual ratios
of dimensions.
Method for Producing Structure Composed of Crosslinked Polymer
First Embodiment
[0036] In one embodiment, the present invention provides a method
for producing a structure composed of crosslinked polymer,
including a step (a) of laminating a photo-dissoluble polymer layer
on a substrate; a step (b) of laminating a layer containing a
crosslinkable polymer on the photo-dissoluble polymer layer; a step
(c) of irradiating the layer containing the crosslinkable polymer
with light in a patternwise manner under crosslinking conditions to
crosslink the crosslinkable polymer in a patternwise manner to
obtain a crosslinked polymer sheet; a step (d) of washing and
removing a uncrosslinked crosslinkable polymer to obtain a
patterned crosslinked polymer sheet; and a step (e) of irradiating
the photo-dissoluble polymer layer with light in a patternwise
manner under dissolution conditions to dissolve the
photo-dissoluble polymer layer in a patternwise mariner, and
peeling the crosslinked polymer sheet in a patternwise manner from
the substrate.
[0037] FIGS. 1A to 1B and 2 are photographs showing specific
examples of structures composed of crosslinked polymer produced by
the production method of the present embodiment. Details of these
structures composed of crosslinked polymer will be described
later.
[0038] FIGS. 3(a) to 3(e) are diagrams showing the production
method of the present embodiment. Hereinafter, the production
method of the present embodiment will be described with reference
to FIG. 3.
[0039] [Step (a)]
[0040] In the present step, a photo-dissoluble polymer layer is
laminated on a substrate.
[0041] (Substrate)
[0042] The substrate can be appropriately selected depending on the
application of the structure composed of crosslinked polymer. For
example, in the case where the structure composed of crosslinked
polymer is used for cell culture or the like, which will be
described later, it preferably has transmittance to light centered
on a wavelength of about 360 to 830 nm so that cells can be
observed with an optical microscope, a fluorescence microscope, or
the like.
[0043] More specific examples of the material for the substrate
include glass and resin. Examples of the resin include an ABS
resin, a polycarbonate resin, a cycloolefin copolymer (COC), a
cycloolefin polymer (COP), an acrylic resin, polyvinyl chloride, a
polystyrene resin, a polyolefin resin such as polyethylene,
polypropylene, or polymethylpentene, polyvinyl acetate,
polyethylene terephthalate (PET), and polyethylene naphthalate
(PEN). These resins may contain various additives, or a plurality
of resins may be mixed.
[0044] The thickness of the substrate is not particularly limited
and it may be, for example about 0.1 to 5 mm, for example about 0.3
to 3 mm, or for example about 0.5 to 1 mm.
[0045] (Photo-Dissoluble Polymer)
[0046] As the photo-dissoluble polymer, for example, a polymer
which can be dissolved in a patternwise manner can be used. For
example, there is a polymer that decomposes by irradiation with
light. Alternatively, there is a polymer in which irradiation with
light in a predetermined solvent leads to isomerization of the
structure without decomposition and the solubility in the solvent
is improved. In the present specification, the term "light" refers
to electromagnetic waves such as ultraviolet light, visible light,
infrared light, X-rays, and .gamma.-rays.
[0047] Examples of the photo-dissoluble polymer include a polymer
containing polyethylene glycol as a main chain and having a
nitrobenzyl group in the molecule; various crosslinked polymers
constituting a positive-type photoresist; a crosslinked polymer
having a photocleavable crosslinking point such as an o-nitrobenzyl
group or a coumarin group; a polymer having a ketone carbonyl group
and a copolymer thereof; a mixed polymer of a ketone copolymer and
other synthetic polymers; a copolymer of ethylene and carbon
monoxide, or a mixed polymer of such a copolymer and other ethylene
polymers; an .alpha.-olefin copolymer containing at least one
photodegradable metal compound which is a metal compound such as
iron, copper, manganese, cobalt, cerium, vanadium, chromium, or
nickel; and a polymer containing acetylacetonate copper and
dialkyldithiocarbamic acid. The photo-dissoluble polymer can be
dissolved, for example, by irradiation with light having a
wavelength of 180 to 600 nm in an appropriate solvent such as
water, a mixed solvent of ethanol and water, or tetramethylammonium
hydroxide.
[0048] The method of laminating a photo-dissoluble polymer layer on
a substrate may be, for example, a method in which a solution in
which the photo-dissoluble polymer is dissolved in an appropriate
solvent is applied onto the substrate by dipping, spraying, spin,
coating, or the like, and then the solvent is removed. The solvent
can be removed by air drying, heating, or the like.
[0049] The thickness of the photo-dissoluble polymer layer is not
particularly limited and it may be, for example about 2 to 1,000
nm, for example about 5 to 500 nm, or for example, about 10 to 200
nm, in a state where the solvent is removed.
[0050] [Step (b)]
[0051] In the present step, a layer containing a crosslinkable
polymer is laminated on the photo-dissoluble polymer layer.
[0052] (Layer Containing Crosslinkable Polymer)
[0053] The layer containing a crosslinkable polymer may be a layer
of a composition containing a polymer capable of crosslinking upon
irradiation with light. Alternatively, the layer containing a
crosslinkable polymer may be a layer of a composition containing a
crosslinking agent capable of crosslinking by the action of an acid
and a crosslinkable polymer. In this case, a polymer layer having a
photoacid generating ability may be further laminated on the layer
containing a crosslinkable polymer. Details will be provided later.
Hereinafter, the term "composition containing a polymer capable of
crosslinking upon irradiation with light" or the term "composition
containing a crosslinking agent capable of crosslinking by the
action of an acid and a crosslinkable polymer" may be referred to
as a "pregel composition" in some cases.
[0054] (Polymer Capable of Crosslinking Upon Irradiation With
Light)
[0055] As the polymer capable of crosslinking upon irradiation with
light, it is possible to use a polymer having a diazirine group, an
azide group, or the like which crosslinks with an amino group or
the like upon irradiation with light.
[0056] (Crosslinking Agent Capable of Crosslinking by Action of
Acid)
[0057] The crosslinking agent capable of crosslinking by the action
of an acid may be, for example, a crosslinking agent containing an
acid labile group capable of forming a carbonium ion in the
presence of a strong acid. A more specific example thereof may be
tetraalkoxymethyl-substituted glycoluril such as tetramethoxymethyl
glycoluril (TMMGU).
[0058] (Crosslinkable Polymer)
[0059] Examples of the crosslinkable polymer include compounds
containing a plurality of hydroxyl groups and having a
weight-average molecular weight of, for example 2,000 or more, for
example 5,000 to 10,000,000, or for example 10,000 to 1,000,000. In
the present specification, the weight-average molecular weight is a
value in terms of standard polyethylene glycol measured by a gel
permeation chromatography (GPC) method. Further, the crosslinkable
polymer may be water-soluble. In the case where the crosslinkable
polymer is water-soluble, it can be a hydrogel having permeability
of low molecular weight components. In addition, the crosslinkable
polymer may be a polysaccharide or a derivative thereof.
[0060] Examples of the polysaccharide or the derivative thereof
include ethylcellulose, methylcellulose, hydroxypropylcellulose,
hydroxymethylcellulose, carboxymethylcellulose, xanthan gum, guar
gum, gum arabic, amylose, agarose, agaropectin, arabinan, curdlan,
callose, carboxymethyl starch, chitin, chitosan, quince seed gum,
glucomannan, gellan gum, tamarind seed gum, dextran, nigeran,
hyaluronic acid, pustulan, funoran, pectin, porphyran, laminaran,
lichenan, carrageenan, alginic acid, tragacanth gum, arukashigamu,
locust bean gum, xanthan gum, rhamsan gum, agar, and
microfibrillated cellulose.
[0061] A more specific example of the crosslinkable polymer may be
a crosslinkable polymer capable of forming a hydrogel in water,
examples thereof include partially saponified polyvinyl acetate,
polyvinyl alcohol, hydroxyalkyl cellulose such as
hydroxypropylcellulose, and a vinyl pyrrolidone-vinyl alcohol
copolymer.
[0062] (Pregel Composition)
[0063] The pregel composition may be, for example, a composition in
which the above-described polymer capable of crosslinking-upon
irradiation with light and, as necessary, an appropriate additive
are dissolved or dispersed in a solvent, or a composition in which
a crosslinking agent capable of crosslinking by the action of an
acid, a crosslinkable polymer and, as necessary, an appropriate
additive are dissolved or dispersed in a solvent. Examples of the
solvent include methanol, ethanol, propanol, and butanol. Examples
of the additive include sulfuric acid, trifluoroacetic acid, and
alkyl sulfuric acid.
[0064] In the case where a composition containing a polymer capable
of crosslinking upon irradiation with light is used as the pregel
composition, the method of laminating the layer of the pregel
composition on the photo-dissoluble polymer layer is not
particularly limited. For example, the pregel composition may be
applied onto the photo-dissoluble polymer layer by dipping,
spraying, spin coating, doctor blade coating, lip coating, or the
like. After applying the pregel composition, it is preferable to
remove the solvent in the pregel composition. The solvent can be
removed by air drying, heating, or the like.
[0065] The thickness of the layer of the pregel composition is not
particularly limited, and it may be, for example about 0.01 to 100
.mu.m, for example about 0.03 to 30 .mu.m, or for example, about
0.1 to 10 .mu.m, in a state where the solvent is removed.
[0066] On the other hand, in the case where a composition
containing a crosslinking agent capable of crosslinking by the
action of an acid and a crosslinkable polymer is used as the pregel
composition, a polymer layer having a photoacid generating ability
may be further laminated on the layer containing a crosslinkable
polymer. This makes it possible for the acid generated by
irradiating the polymer layer having a photoacid generating ability
with light to change the crosslinking agent capable of crosslinking
by the action of an acid into a state capable of crosslinking, so
that it becomes possible to crosslink the crosslinkable polymer.
That is, by laminating a polymer layer having a photoacid
generating ability on a layer containing a crosslinkable polymer,
it is possible to crosslink the crosslinkable polymer in
patternwise manner through patternwise light irradiation. Here, the
photon-dissoluble polymer layer may have a photoacid generating
ability. That is, a photo-dissoluble polymer layer having a
photoacid generating ability may be used as the photo-dissoluble
polymer layer.
[0067] (Polymer Having Photoacid Generating Ability)
[0068] As the polymer having a photoacid generating ability, for
example, a polymer capable of generating an acid in a patternwise
manner can be used. For example, a polymer capable of generating an
acid upon irradiation with light can be mentioned. Examples of such
a polymer include those having a structure consisting of a
chromophore absorbs light and an acid precursor that becomes an
acidic substance after decomposition.
[0069] As the polymer having a photoacid generating ability, for
example, a polymer containing a photoacid generator can be
used.
[0070] Examples of the photoacid generator include sulfonic acid
derivatives, carboxylic acid esters, and onium salts.
[0071] Examples of the sulfonic acid derivatives include a
naphthalene imide-based sulfonic acid derivative and a
thioxanthone-based sulfonic acid derivative. The naphthalene
imide-based sulfonic acid derivative may be, for example, sulfonic
acid 1,8-naphthalimide. The thioxanthone-based sulfonic acid
derivative may be, for example, sulfonic acid
1,3,6-trioxo-3,6-dihydro-1H-11-thia-azacyclopenta[a]anthracen-2-yl
ester. Examples of other sulfonic acid derivatives include
disulfones, disulfonyldiazomethanes, disulfonylmethanes,
sulfonylbenzoylmethanes, imide sulfonates, and benzoin
sulfonates.
[0072] Examples of the carboxylic acid esters include
1,8-naphthalenedicarboxylic acid imide methylsulfonate and
1,8-naphthalenedicarboxylic acid imide tosylsulfonate.
[0073] Examples of the onium salts include sulfonium salts and
iodonium salts having an anion such as tetrafluoroborate
(BF.sub.4.sup.-), hexafluorophosphate (PF.sub.6.sup.-), or
hexafluoroantimonate (SbF.sub.6.sup.-).
[0074] Examples of the polymer include a polystyrene-based resin
and a (meth)acrylic resin. In the present specification, the
(meth)acrylic resin refers to an acrylic resin and a methacrylie
resin.
[0075] The method of laminating a polymer layer having a photoacid
generating ability on a substrate is the same as the
above-described method of laminating a photo-dissoluble polymer
layer on a substrate.
[0076] The thickness of the polymer layer having a photoacid
generating ability is not particularly limited and it may be, for
example about 2 to 1,000 nm, for example about 5 to 500 nm, or for
example, about 10 to 200 nm, in a state where the solvent is
removed.
[0077] (Photo-Dissoluble Polymer Having Photoacid Generating
Ability)
[0078] A photo-dissoluble polymer having a photoacid generating
ability may be used in place of the photo-dissoluble polymer and
the polymer having a photoacid generating ability described above.
More specific examples of the photo-dissoluble polymer having a
photoacid generating ability include polymethyl methacrylate having
a photoacid generating group in the side chain (hereinafter,
sometimes referred to as "pPAGMMA"), polymethyl methacrylate, and
poly(N-alkyl acrylamide). Examples of the photoacid generating
group include a sulfonate derivative such as naphthalene
imide-based sulfonic acid or thioxanthone-based sulfonic acid, a
sulfonyl compound, and an onium compound.
[0079] The pPAGMMA is capable of generating protons upon
irradiation with light having a wavelength of 320 to 480 nm in the
atmosphere or the like environment. Further, the pPAGMMA may be
dissolved in an appropriate solvent such as ethanol containing 20
mass % of water, upon irradiation with light having a wavelength of
320 to 480 nm.
[0080] The method of laminating a photo-dissoluble polymer layer
having a photoacid generating ability on a substrate is the same as
the above-described method of laminating a photo-dissoluble polymer
layer on a substrate.
[0081] The thickness of the photo-dissoluble polymer layer having a
photoacid generating ability is not particularly limited and it may
be, for example about 2 to 1,000 nm, for example about 5 to 500 nm,
or for example about 10 to 200 nm, in a state where the solvent is
removed.
[0082] In the case where a composition containing a crosslinking
agent capable of crosslinking by the action of an acid and a
crosslinkable polymer is used as the pregel composition, the method
of applying the pregel composition on the polymer layer having a
photoacid generating ability or the photo-dissoluble polymer layer
having a photoacid generating ability is not particularly limited.
For example, there is a method in which the pregel composition is
applied onto the polymer layer having a photoacid generating
ability or the photo-dissoluble polymer layer having a photoacid
generating ability by dipping, spraying, spin coating, doctor blade
coating, lip coating, or the like. After applying the pregel
composition, it is preferable to remove the solvent in the pregel
composition. The solvent can be removed by air drying, heating, or
the like.
[0083] The thickness of the layer of the pregel composition is the
same as in the case of the above-described composition containing a
polymer capable of crosslinking upon irradiation with light.
[0084] FIG. 3(a) is a diagram showing a state in which a
photo-dissoluble polymer layer 20 having a photoacid generating
ability is laminated on a substrate 10 and a pregel composition (a
composition containing a crosslinking agent capable of crosslinking
by the action of an acid and a crosslinkable polymer) 30 is further
applied thereon, as an example.
[0085] [Step (c)]
[0086] In the present step, the layer containing a crosslinkable
polymer is irradiated with light in a patternwise manner under
crosslinking conditions to crosslink the crosslinkable polymer in a
patternwise manner, thereby obtaining a crosslinked polymer
sheet.
[0087] (Light Irradiation Under Crosslinking Conditions)
[0088] In the case where the above-described composition containing
a polymer capable of crosslinking upon irradiation with light is
used as the pregel composition, the term "under crosslinking
conditions" refers to appropriate conditions capable of
crosslinking the polymer used. The crosslinkable polymer in the
pregel composition is crosslinked upon irradiation with light.
[0089] As a result, the pregel composition forms a crosslinked
polymer sheet in the portion irradiated with light.
[0090] In the case where the above-described composition containing
a crosslinking agent capable of crosslinking by the action of an
acid and a crosslinkable polymer is used as the pregel composition,
the term "under crosslinking conditions" refers to appropriate
conditions corresponding to the polymer having a photoacid
generating ability or the photo-dissoluble polymer having a
photoacid generating ability used. For example, in the case where
the above-described pPAGMMA is used as the photo-dissoluble polymer
having a photoacid generating ability, the crosslinking conditions
may be, for example, conditions such as irradiating 3 J/cm.sup.2 of
light having a wavelength of 436 nm in the air. Light irradiation
in a patternwise manner may be carried out by a general
photolithography technique.
[0091] Irradiation of light leads to generation of an acid in the
polymer layer having a photoacid generating ability or the
photo-dissoluble polymer layer having a photoacid generating
ability, whereby the crosslinkable polymer is crosslinked by the
crosslinking agent in the pregel composition. As a result, the
pregel composition forms a crosslinked polymer sheet in the portion
irradiated with light. After light irradiation, heating or the like
may be carried out to accelerate the crosslinking of the
crosslinkable polymer.
[0092] FIG 3(b) is a diagram showing a state in which the
photo-dissoluble polymer layer 20 having a photoacid generating
ability is irradiated with light in a patternwise manner to
crosslink the crosslinkable polymer.
[0093] [Step (d)]
[0094] In the present step, the crosslinkable polymer which has not
been crosslinked is washed and removed to obtain a patterned
crosslinked polymer sheet. For washing, an appropriate washing
liquid can be used. The washing liquid may be, for example, ethanol
containing water. FIG. 3(c) is a diagram showing a state in which
the pregel composition of the portion not irradiated with light is
washed and removed. A crosslinked polymer sheet 31 is formed in the
portion irradiated with light.
[0095] [Step (e)]
[0096] In the present step, the photo-dissoluble polymer layer or
the photo-dissoluble polymer layer having a photoacid generating
ability is irradiated with light in a patternwise manner under
dissolution conditions to dissolve the photo-dissoluble polymer
layer or the photo-dissoluble polymer layer having a photoacid
generating ability in a patternwise manner, and the crosslinked
polymer sheet is peeled in a patternwise manner from the
substrate.
[0097] (Light Irradiation Under Dissolution Conditions)
[0098] The dissolution conditions refer to appropriate conditions
corresponding to the photo-dissoluble polymer or the
photo-dissoluble polymer having a photoacid generating ability
used. For example, in the case where the above-described pPAGMMA is
used as the photo-dissoluble polymer having a photoacid generating
ability, the dissolution conditions may be, for example, conditions
such as irradiating 10 J/cm.sup.2 of light having a wavelength of
436 nm in a mixed solvent of ethanol and water. Light irradiation
in a patternwise manner may be carried out by a general
photolithography technique.
[0099] Irradiation of light leads to dissolution of the
photo-dissoluble polymer or the photo-dissoluble polymer having a
photoacid generating ability. As a result, in the portion
irradiated with light, the crosslinked polymer sheet described
above is peeled from the substrate. FIG. 3(d) is a diagram showing
a state in which the photo-dissoluble polymer layer 20 having a
photoacid generating ability is irradiated with light in a
patternwise manner in a suitable solvent 40 to dissolve the
photo-dissoluble polymer having a photoacid generating ability.
FIG. 3(e) is a diagram showing a state in which the crosslinked
polymer sheet 31 is peeled off in a patternwise manner from the
substrate 10, as a result of dissolution of the photo-dissoluble
polymer having a photoacid generating ability. In the example of
FIG. 3(e), a pocket-like structure composed of crosslinked polymer
is formed. The pocket-like structure composed of crosslinked
polymer will be described later.
[0100] According to the production method of the present
embodiment, by appropriately setting a pattern for crosslinking the
pregel composition and a pattern for dissolving the
photo-dissoluble polymer or the photo-dissoluble polymer having a
photoacid generating ability, structures composed of crosslinked
polymer of various shapes can be produced. The time from selection
of the pattern of light irradiation to completion of the structure
composed of crosslinked polymer is only a few minutes.
[0101] Since the production method of the present embodiment is
extremely simple, it is possible to provide a powerful experiment
examination means at the research site of cell engineering, or the
like, where the importance of a pattern culture system has been
widely recognized.
Second Embodiment
[0102] In one embodiment, the present invention provides a method
for producing a structure composed of crosslinked polymer further
including a step (f) of breaking and removing the crosslinked
polymer sheet peeled off in a patternwise manner from the
substrate, in addition to the steps (a) to (e) in the
above-described first embodiment. The breakage and removal of the
crosslinked polymer sheet may be carried out, for example, by
applying a physical force to the crosslinked polymer sheet.
[0103] [Step (f)]
[0104] in the present step, the crosslinked polymer sheet peeled
off in a patternwise manner from the substrate is broken and
removed in a patternwise manner.
[0105] The method for applying a physical force may be, for
example, strongly spraying a liquid such as water with an automatic
pipetter or the like which is used in cell culture.
[0106] FIG. 3(f) is a diagram showing a manner of breaking and
removing the crosslinked polymer sheet 31 peeled off in a
patternwise manner from the substrate. For example, as shown by the
arrow in FIG. 3(f) the crosslinked polymer sheet 31 peeled off from
the substrate is broken and removed by spraying a liquid 40 on the
pocket-like structure composed of crosslinked polymer in a suitable
liquid 40.
[0107] According to the production method of the present
embodiment, a region where the substrate is substantially exposed
and a region where the polymer sheet is adhered on the substrate
can be formed in a patternwise manner. Here, the region where the
substrate is substantially exposed means that a state in which the
photo-dissoluble polymer layer or the photo-dissoluble polymer
layer having a photoacid generating ability is not completely
dissolved but remains slightly in the production method of the
present embodiment is also included in the region where the
substrate is exposed.
[0108] The structure composed of crosslinked polymer produced by
the production method of the present embodiment can be used, for
example, as a polymer structure for cell culture. The structure
composed of crosslinked polymer for cell culture will be described
later.
Laminate for Producing Structure Composed of Crosslinked
Polymer
[0109] In one embodiment, the present invention provides a laminate
for producing a structure composed of crosslinked polymer,
including a substrate, a photo-dissoluble polymer layer laminated
on the substrate, and a layer containing a crosslinkable polymer
laminated on the photo-dissoluble polymer layer.
[0110] The laminate for producing a structure composed of
crosslinked polymer of the present embodiment may be one including
a substrate, a photo-dissoluble polymer layer laminated on the
substrate, and a polymer layer having a photoacid generating
ability laminated on the photo-dissoluble polymer layer, or a
photo-dissoluble polymer layer having a photoacid generating
ability laminated on the substrate; and a composition containing a
crosslinking agent capable of crosslinking by the action of an acid
and a crosslinkable polymer, coated on the polymer layer having a
photoacid generating ability or the photo-dissoluble polymer layer
having a photoacid generating ability.
[0111] It is possible to produce various structures composed of
crosslinked polymer by carrying out the steps (c) to (e) of the
above-described method for producing a structure composed of
crosslinked polymer on the laminate for producing a structure
composed of crosslinked polymer of the present embodiment.
[0112] In the laminate for producing a structure composed of
crosslinked polymer of the present embodiment, as for the
substrate, the photo-dissoluble polymer, the crosslinkable polymer,
the composition (pregel composition) containing a polymer capable
of crosslinking upon irradiation with light, the polymer having a
photoacid generating ability, the photo-dissoluble polymer having a
photoacid generating ability, the crosslinking agent capable of
crosslinking by the action of an acid, and the composition (pregel
composition) containing a crosslinking agent capable of
crosslinking by the action of an acid and a crosslinkable polymer,
the same ones as those described above can be used.
Structure Composed of Crosslinked Polymer
First Embodiment
[0113] In one embodiment, the present invention provides a
structure composed of crosslinked polymer including a substrate and
a crosslinked polymer sheet provided on the substrate, in which the
substrate and the crosslinked polymer sheet have an adhesive region
where the substrate and the crosslinked polymer sheet are adhered,
and a non-adhesive region where the substrate and the crosslinked
polymer sheet are not adhered, in a planar view, the adhesive
region of the substrate is closed to surround the periphery of the
non-adhesive region of the substrate, and the non-adhesive region
of the crosslinked polymer sheet provided with at least one
through-hole. In the present specification, the term "planar view"
refers to a state in which the substrate is viewed from a direction
perpendicular to the substrate.
[0114] The structure composed of crosslinked polymer of the present
embodiment can be produced, for example, by the method for
producing a structure composed of crosslinked polymer according to
the above-described first embodiment. Therefore, the substrate, the
photo-dissoluble polymer, the crosslinkable polymer, the
composition (pregel composition) containing a polymer capable of
crosslinking upon irradiation with light, the polymer having a
photoacid generating ability, the photo-dissoluble polymer having a
photoacid generating ability, the crosslinking agent capable of
crosslinking by the action of an acid, and the composition (pregel
composition) containing a crosslinking agent capable of
crosslinking by the action of an acid and a crosslinkable polymer,
which are used for producing the structure composed of crosslinked
polymer of the present embodiment, are the same as those used in
the method for producing a structure composed of crosslinked
polymer according to the above-described first embodiment.
[0115] In the structure composed of crosslinked polymer of the
present embodiment, the adhesive region to which the substrate and
the polymer sheet are adhered refers to a region where the
crosslinked polymer sheet was not peeled off from the substrate in
the step (e), in the region where the pregel composition was
crosslinked to form a crosslinked polymer sheet in step (c) of the
method for producing a structure composed of crosslinked polymer
according to the above-described first embodiment.
[0116] Here, the adhesive region of the substrate refers to the
above-described adhesive region in the substrate, and the adhesive
region of the crosslinked polymer sheet refers to the
above-described adhesive region in the crosslinked polymer
sheet.
[0117] In addition, the non-adhesive region refers to a region
where the crosslinked polymer sheet was peeled off from the
substrate in the step (e), in the region where the pregel
composition was crosslinked to form a crosslinked polymer sheet in
the step (c) of the method for producing a structure composed of
crosslinked polymer according to the above-described first
embodiment.
[0118] Here, the non-adhesive region of the substrate refers to the
above-described non-adhesive region in the substrate (region where
the substrate is substantially exposed), and the non-adhesive
region of the crosslinked polymer sheet refers to the
above-described non-adhesive region in the crosslinked polymer
sheet (region of the crosslinked polymer sheet which is peeled from
the substrate).
[0119] In the structure composed of crosslinked polymer of the
present embodiment, in a planar view, the adhesive region of the
substrate is closed to surround the periphery of the non-adhesive
region of the substrate. In addition, at least one through-hole
exists in the non-adhesive region of the crosslinked polymer sheet.
As a result, the structure composed of crosslinked polymer of the
present embodiment forms a pocket-like structure (bag-like
structure).
[0120] Specific examples of the structure composed of crosslinked
polymer of the present embodiment include the structures shown in
FIGS. 1(a) to 1(e) and 2.
[0121] For example, in the structure shown in FIGS. 1A, 1B and 2,
it can be said that the lower through-hole (opening) is the
entrance of the pocket. Further, in the structures shown in FIGS.
1C, 1D and 1E, it can be said that the front side of the substrate
or the back side of the substrate is the entrance of the
pocket.
[0122] The pocket-like structure composed of crosslinked polymer
may have any type of opening. The crosslinked polymer sheet
constituting the pocket-like structure composed of crosslinked
polymer may have one or a plurality of through-holes in addition to
the opening of the pocket. For example, in the structure shown in
FIG. 1A, three through-holes are formed on the upper side of the
structure. In the structures shown in FIGS. 1B and 1D and in the
right half of FIG. 2 (the third and fourth rows from the left), a
plurality of through-holes are formed in the crosslinked polymer
sheet to form a mesh-like structure. Also in the structure shown in
FIG. 1E, a plurality of through-holes are formed in the crosslinked
polymer sheet.
[0123] (Application of Structure Composed of Crosslinked Polymer of
First Embodiment)
[0124] Cell Culture
[0125] While utilization of human iPS-derived cells is being
studied seriously, techniques for culturing cells adhered on a
culture substrate in a flowing culture fluid (perfusion culture)
have been extensively investigated, particularly in the field of
pharmaceutical assay or the like.
[0126] In perfusion culture in the field of pharmaceutical assay or
the like, it is important that cells are immobilized on a substrate
without floating, so that the cells can be observed and evaluated
at any time. In many cases, anchorage-dependent cells are
previously adhered on the substrate and then the culture fluid is
allowed to flow. However, since it is impossible to allow the
culture fluid to flow for the time until the cells are adhered on
the substrate, depletion of oxygen and nutrients may be a
problem.
[0127] In addition, in order to carry out perfusion culture of
floating cells and cell masses having no adhesiveness on a
substrate that can be monitored at any time, it is necessary to
have a mechanism for immobilizing them. Application of a material
having a minute structure on the surface has been studied as a
technique for keeping a floating micro object at a predetermined
position in a flowing liquid. For example, using a columnar
structure disposed in a flow path has been studied.
[0128] However, in the case where floating cells or cell masses
having no adhesiveness are held in a predetermined region by means
of a columnar structure disposed in the flow path, there may be a
case where the flow of the culture fluid is deteriorated due to the
cells blocked by the columnar structure. As a result, the original
purpose of perfusion culture to spread a fresh culture fluid to
cells cannot be achieved.
[0129] On the other hand, by capturing floating cells or cell
masses having no adhesiveness inside the above-described
pocket-like structure composed of crosslinked polymer, it is
possible to keep the cells in a predetermined region. This makes it
possible to flow a culture medium immediately after capturing the
cells inside the pocket structure. At that time, the culture fluid
can flow freely above the pocket structure, and there is no
disturbance of the overall flow. In addition, since the crosslinked
polymer sheet constituting the pocket is thin and transparent, it
does not seriously affect the observation of the cells inside. In
addition, since the pocket portion can be broken by strongly
spraying the culture fluid to the pocket structure, the cells can
be easily recovered from the inside of the pocket structure.
[0130] Further, by providing a plurality of through-holes in the
crosslinked polymer sheet constituting the pocket, an object having
a size larger than the size of the through-hole is captured while
an object or fluid having a size equal to or smaller than the size
of the through-hole can pass through the interior of the pocket.
Therefore, it is also possible to selectively capture an object of
a predetermined size and efficiently separate it from an object
having a size equal to or smaller than a predetermined size. In so
doing, the fluid can be supplied to the captured object surface
without its flow being disturbed by the pocket structure. That is,
a useful cell perfusion culture means can be provided by capturing
floating cells or cell masses having no adhesiveness as the
above-described object in the above-described pocket-like structure
composed of crosslinked polymer.
[0131] In the case where the structure composed of crosslinked
polymer of the first embodiment is used for cell culture, the area
of the non-adhesive region of the substrate may be, for example 10
to 100,000,000 .mu.m.sup.2, for example 50 to 10,000,000
.mu.m.sup.2, or for example, 100 to 1,000,000 .mu.m.sup.2.
[0132] Valve
[0133] Also, the pocket-like structure composed of crosslinked
polymer is inflated in response to the flow of a fluid from the
inlet direction of the pocket, thus blocking the flow of the fluid,
while it is deflated in response to the flow of the fluid in the
opposite direction, thus not blocking the flow of the fluid.
Therefore, by arranging the pocket-like structure composed of
crosslinked polymer in the flow path, it is possible to form a flow
path in which the flow path resistance varies depending on the
direction of the fluid flow. Thus, the pocket-like structure
composed of crosslinked polymer may serve to function as a valve,
for example, in a fluidic device.
Second Embodiment
[0134] In one embodiment the present invention provides a structure
composed of crosslinked polymer for cell culture, including: a
substrate; and a cell adhesivable portion and a non-cell
adhesivable portion provided in a patternwise manner on the
substrate, in which the substrate is exposed in the cell
adhesivable portion, and the non-cell adhesivable portion is made
of a crosslinked polymer sheet adhered on the substrate. The
structure composed of crosslinked polymer for cell culture of the
present embodiment can also be said to be a cell culture
substrate.
[0135] The structure composed of crosslinked polymer of the present
embodiment can be produced, for example, by the method for
producing a structure composed of crosslinked polymer according to
the above-described second embodiment. Therefore, the substrate,
the photo-dissoluble polymer, the crosslinkable polymer, the
composition (pregel composition) containing a polymer capable of
crosslinking upon irradiation with light, the polymer having a
photoacid generating ability, the photo-dissoluble polymer having a
photoacid generating ability, the crosslinking agent capable of
crosslinking by the action of an acid, and the composition pregel
composition) containing a crosslinking agent capable of
crosslinking by the action of an acid and a crosslinkable polymer,
which are used for producing the structure composed of crosslinked
polymer of the present embodiment, are the same as those used in
the method for producing a structure composed of crosslinked
polymer according to the above-described second embodiment.
[0136] In the structure composed of crosslinked polymer of the
present embodiment, cells can adhere to the cell adhesivable
portion. On the other hand, the non-cell adhesivable portion has a
strong cell adhesion inhibitory property, so that cell adhesion is
strongly inhibited. By using the structure composed of crosslinked
polymer of the present embodiment, it is possible to construct a
pattern culture system in which cells are adhered in a patternwise
manner and cultured.
[0137] In the structure composed of crosslinked polymer of the
present embodiment, the cell adhesivable portion is a region in
which the substrate is substantially exposed. In addition, the
non-cell adhesivable portion is a region where the crosslinked
polymer sheet is adhered to the substrate. In particular, since a
hydrogel made of a neutral hydratable polymer has a strong cell
adhesion inhibitory property, it is suitable as a material
constituting the non-cell adhesivable portion of the structure
composed of crosslinked polymer of the present embodiment.
[0138] (Arrangement Example 1 of Cell Adhesivable Portion and
Non-Cell Adhesivable Portion)
[0139] In the structure composed of crosslinked polymer for cell
culture of the present embodiment, the cell adhesivable portion and
the non-cell adhesivable portion may be linearly arranged in
parallel with each other.
[0140] As shown in Examples to be described later, FIG. 7A is a
photograph showing the results of culturing cells on a structure
composed of crosslinked polymer for cell culture in which a cell
adhesivable portion and a non-cell adhesivable portion are linearly
arranged in parallel with each other. By arranging the cell
adhesivable portion and the non-cell adhesivable portion in this
way, cells can be aligned and oriented in parallel.
[0141] (Arrangement Example 2 of Cell Adhesivable Portion and
Non-Cell Adhesivable Portion)
[0142] In the structure composed of crosslinked polymer for cell
culture of the present embodiment, the cell adhesivable portion may
have a plurality of island-like portions and connecting portions
for connecting together the plurality of the island-like
portions.
[0143] The present inventors have found that, in the case where
cells are cultured using a structure composed of crosslinked
polymer for cell culture having a plurality of independent
island-like cell adhesivable portions, the degree of proliferation
of cells in each of the island-like portions tends to be
non-uniform.
[0144] The present inventors further have found that, in the case
where cells are cultured using a structure composed of crosslinked
polymer for cell culture in which a pattern shape of a cell
adhesivable portion is formed into a shape having a plurality of
independent island-like portions and connecting portions for
connecting together the island-like portions, the degree of
proliferation of cells in each of the island-like portions tends to
be uniform.
[0145] That is, by using a structure composed of crosslinked
polymer for cell culture having a cell adhesivable portion, having
a shape having a plurality of island-like portions and connecting
portions for connecting together the plurality of the island-like
portions, the degree of proliferation of the cells in each of the
island-like portions can be made uniform and the cells can be
homogeneously cultured.
[0146] As shown in Examples to be described later, FIGS. 6, 7B, and
8 are photographs showing the results of culturing cells on a
structure composed of crosslinked polymer for cell culture in which
the cell adhesivable portion has a plurality of island-like
portions and connecting portions for connecting together the
plurality of the island-like portions. By arranging the cell
adhesivable portion and the non-cell adhesivable portion in this
way, the degree of proliferation of the cells in each of the
island-like portions can be made uniform and the cells can be
homogeneously cultured.
[0147] In the structure composed of crosslinked polymer for cell
culture of the present embodiment, the shape of the island-like
portion is not particularly limited, and examples thereof include a
circular shape, a rectangular shape, a polygonal shape, and other
indefinite shapes. Here, the circular shape includes an elliptical
shape. The polygonal shape may be a triangular shape, a
quadrangular shape, a pentagonal shape, a hexagonal shape, or the
like.
[0148] Further, it can be said that the portion of the cell
adhesivable portion that is narrow in the top view is the
connecting portion and the portion excluding the connecting portion
is the island-like portion.
[0149] The area per island-like portion may be, for example 200 to
100,000,000 .mu.m.sup.2, for example 1,000 to 200,000 .mu.m.sup.2,
or for example 5,000 to 50,000 .mu.m.sup.2.
[0150] In addition, in the case where it is intended to produce a
cell mass having a uniform size as described below, it is
preferable that all of the island-like portions have substantially
the same area. Here, the term "substantially the same" is intended
to permit variations that are difficult to eliminate in the
production process.
Cell Culture Method and Cells
[0151] In one embodiment the present invention provides a cell
culture method for uniformly culturing cells, including a step of
culturing cells on a surface of a structure composed of crosslinked
polymer for cell culture in which a cell adhesivable portion has a
plurality of island-like portions and connecting portions for
connecting together the plurality of the island-like portions, and
all of the island-like portions have substantially the same area,
with the cells being adhered to the plurality of the island-like
portions.
[0152] As described above, the present inventors found that, by
culturing cells on the surface of such a structure composed of
crosslinked polymer for cell culture, the cells adhered to the
plurality of the island-like portions can be uniformly
cultured.
[0153] In one embodiment, the present invention provides a cell
cultured by the cell culture method described above.
[0154] The cell of the present embodiment may be distributed
together with a structure composed of crosslinked polymer for cell
culture in a state of the cell being adhered on the structure
composed of crosslinked polymer for cell culture.
Method For Producing Cell Mass and Cell Mass
[0155] In one embodiment, the present invention provides a method
for producing a cell mass, including a step of culturing cells on a
surface of a structure composed of crosslinked polymer for cell
culture in which a cell adhesivable portion has a plurality of
island-like portions and connecting portions for connecting
together the plurality of the island-like portions, and all of the
island-like portions have substantially the same area; and a step
of detaching the cells to obtain substantially the same number of
cell masses as the number of the island-like portions consisting of
the cells adhering to the plurality of the island-like portions.
According to the method for producing a cell mass of the present
embodiment, it is possible to obtain a cell mass having a uniform
size.
[0156] Here, the phrase "substantially the same number of cell
masses as the number of the island-like portions" is intended to
permit there is a case where the number of cell masses completely
matched with the number of the island-like portions cannot be
obtained due to destruction of a cell mass or the like which cannot
be excluded in operations such as detachment of cells.
[0157] For example, in research using iPS cells or iPS-derived
cells, or in fields utilizing these cells, it is sometimes
necessary to prepare a uniform cell mass of these cells. According
to the method for producing a cell mass of the present embodiment,
it is possible to easily produce a cell mass of iPS cells or the
like having a uniform size.
[0158] The method for detaching the cells may be, for example, a
method of reacting the cells with an enzyme such as trypsin or
collagenase, or a method of washing the cells with a buffer
solution containing a chelating agent to remove calcium ions
necessary for cell adhesion.
[0159] In one embodiment, the present invention provides a cell
mass cultured by the above-described method for producing a cell
mass.
[0160] Since the cell mass of the present embodiment exhibits a
uniform size, it can be effectively used for, for example, cell
assays of pharmaceuticals and chemical products, and research and
utilization using iPS cells.
EXAMPLES
[0161] Hereinafter, the present invention will be described in more
detail with reference to the following Examples, but the present
invention is not limited thereto.
Example 1
Formation Of Pocket-Like Structure Composed of Crosslinked Polymer
1
[0162] pPAGMMA containing a photoacid generating residue and having
a monomer fraction of 2 mol % was used as a photo-dissoluble
polymer having a photoacid generating ability. First, a
trifluoroethanol solution containing 1 mass % of pPAGMMA was
spin-coated on a polystyrene substrate and heated at 85.degree. C.
for 1 hour.
[0163] Subsequently, a methanol solution containing 5 mass % of
partially hydrolyzed polyvinyl acetate, 0.1mass % of
tetramethoxymethylglycoluril (hereinafter, sometimes referred to as
"TMMGU" ), and 0.005 mass % of sulfuric acid was prepared as a
composition containing a crosslinking agent capable of crosslinking
by the action of an acid and a crosslinkable polymer. The
composition was spin-coated on the substrate and heated at
85.degree. C. for 5 minutes.
[0164] This was followed by exposure to light having a wavelength
of 436 nm at an irradiation dose of 3 J/cm.sup.2 along the pattern
forming a polymer sheet (gel layer). Subsequently, after heating at
85.degree. C. for 2 hours, development was carried out by washing
and removing the unexposed portion with ethanol containing
water.
[0165] The formed polymer sheet was exposed to light having a
wavelength of 436 nm at an irradiation dose of 10 J/cm.sup.2 in a
patternwise manner in ethanol containing 20 weight % of water to
dissolve pPAGMMA, and the polymer sheet was locally peeled off from
the substrate surface to form a pocket-like structure composed of
crosslinked polymer.
Example 2
Formation of Pocket-Like Structure Composed of Crosslinked Polymer
2
[0166] pPAGMMA containing a photoacid generating residue and having
a monomer fraction of 2 mol % was used as a photo-dissoluble
polymer having a photoacid generating ability. First, a
trifluoroethanol solution containing 1 mass % of pPAGMMA was
spin-coated on a polystyrene substrate and heated at 85.degree. C.
for 1 hour.
[0167] Subsequently, a methanol solution containing 5 mass % of
hydroxypropylcellulose, 0.1 mass % of TMMGU, and 0.005 mass % of
sulfuric acid was prepared as a composition containing a
crosslinking agent capable of crosslinking by the action of an acid
and a crosslinkable polymer. The composition was spin-coated on the
substrate and heated at 85.degree. C. for 5 minutes.
[0168] This was followed by exposure to light having a wavelength
of 436 nm at an irradiation dose of 3 J/cm.sup.2 along the pattern
forming a polymer sheet. Subsequently, after heating at 85.degree.
C. for 2 hours, development was carried out by washing and removing
the unexposed portion with water.
[0169] The formed polymer sheet was exposed to light having a
wavelength of 436 nm at an irradiation dose of 10 J/cm.sup.2 in a
patternwise manner in ethanol containing 20 weight % of water to
dissolve pPAGMMA, and the polymer sheet was locally peeled off from
the substrate surface to form a pocket-like structure composed of
crosslinked polymer having one or a plurality of through-holes in
the peeled area of the polymer sheet.
[0170] FIGS. 1A to 1E are photographs showing the results of
confocal laser scanning microscopic observation of a pocket-like
structure composed of crosslinked polymer formed in the present
Example. FIG. 2 is a photograph showing the results of optical
microscopic observation of a pocket array in which pocket-like
structures composed of crosslinked polymer are arranged in an
array, which was formed in the present Example. In the pocket-like
structure composed of crosslinked polymer shown in the right half
of FIG. 2, a large number of rectangular through-holes are formed
in a region where the polymer sheet is peeled off (a region where
the pocket is formed) and are in a mesh shape (hereinafter,
sometimes referred to as "mesh-like pocket structure").
Example 3
Cell Culture Using Pocket-Like Structure Composed of Cross Linked
Polymer
[0171] With respect to a substrate having a large number of
pocket-like structures composed of crosslinked polymer formed in
Example 2 on the surface thereof, cells were introduced into the
pocket structure by repeatedly pouring the cell dispersion of MDCK
cells, a cell line derived from canine renal tubule epithelial
cells, or HepG2 cells, a cell line derived from human liver cancer,
dispersed in a medium from the inlet direction of the pocket. The
cells were cultured in an incubator as they were. FIG. 4 is a
photograph showing the results of optical microscopic observation
of the MDCK cells cultured in the present Example. As a result, it
was confirmed that the cells were stably retained and survived in
the pocket structure even after the next day.
Example4
Cell Separation Using Pocket Structure
[0172] With respect to a substrate having a large number of
mesh-like pocket structures formed in Example 2 on the surface
thereof, a cell mass was introduced into the pocket structure by
repeatedly pouring the cell dispersion containing the cell mass of
human iPS cells having a distribution in size from the inlet
direction of the pocket. Furthermore, it was confirmed that only
the cell mass larger than the mesh structure of the pocket can be
left inside the pocket structure by tilting the substrate with the
inlet direction of the pocket facing upward and pouring the medium
from the pocket inlet direction. The cells were cultured in an
incubator as they were. FIGS. 5A and 5B are photographs showing the
results of optical microscopic observation of the cell mass of
human iPS cells cultured in the present Example. As a result, it
was confirmed that the cells were stably retained and survived in
the pocket structure even after the next day.
Example 5
Preparation of Structure Composed of Crosslinked Polymer for Cell
Culture
[0173] pPAGMMA containing a photoacid generating residue and having
a monomer fraction of 2 mol % was used as a photo-dissoluble
polymer having a photoacid generating ability. First, a
trifluoroethanol solution containing 0.5 mass % of pPAGMMA was
spin-coated on a polystyrene substrate and heated at 85.degree. C.
for 1 hour.
[0174] Subsequently, a methanol solution containing 0.1 mass % of
hydroxypropylcellulose, 0.001 mass % of TMMGU, and 0.002 mass % of
sulfuric acid was prepared as a composition containing a
crosslinking agent capable of crosslinking by the action of an acid
and a crosslinkable polymer. The composition was spin-coated on the
substrate and heated at 85.degree. C. for 5 minutes.
[0175] This was followed by exposure of the entire surface of the
substrate to light having a wavelength of 436 nm at an irradiation
dose of 3 J/cm.sup.2. Subsequently, the substrate was heated at
85.degree. C. for 2 hours. As a result, a polymer sheet was formed
on the entire surface of the substrate.
[0176] Subsequently, the formed polymer sheet was exposed to light
having a wavelength of 436 nm at an irradiation dose of 10 J/cm2 in
a patternwise manner in ethanol containing 20 weight % of water to
dissolve pPAGMMA, and the polymer sheet was locally peeled off from
the substrate surface. Subsequently, the substrate surface was
intensively washed with water, whereby the peeled polymer sheet was
broken and removed in a patternwise manner.
[0177] In this manner, the structure composed of crosslinked
polymer for cell culture having a cell adhesivable portion and a
non-cell adhesivable portion was obtained. Here, the region from
which the polymer sheet has been removed is the cell adhesivable
portion, and the portion where the polymer sheet is present is the
non-cell adhesivable portion.
[0178] MDCK cells, NIH/3T3 cells, a mouse fetus-derived fibroblast
cell line, or human iPS cells were seeded on the resulting
structure composed of crosslinked polymer for cell culture and
cultured until the next day. In the culture of human iPS cells, the
surface of the structure composed of crosslinked polymer for cell
culture was coated with Matrigel (manufactured by Corning
Incorporated).
[0179] As a result, in any of the cells, the cells adhered only to
the region from which the polymer sheet was removed (the cell
adhesivable portion), and therefore the cells could be cultured in
a patternwise manner. In addition, the cell adhesion inhibition of
the region where the polymer sheet is present (non-cell adhesivable
portion) was very high, and the cells grown to overconfluence by
continuous culture for another 2 days also did not protrude from
the non-cell adhesivable portion.
[0180] FIG. 6 is a photograph showing the results of optical
microscopic observation of the MDCK cells cultured in the present
Example. In the case where the cells were detached in this state,
the cells in each of the island-like cell adhesivable portions
formed one cell mass each, and therefore a cell mass having a
uniform size could be obtained.
[0181] FIGS. 7A and 7B are photographs showing the results of
optical microscopic observation of the NIH/3T3 cells cultured in
the present Example. In the structure composed of crosslinked
polymer for cell culture shown in FIG. 7A, the cell adhesivable
portion and the non-cell adhesivable portion were linearly arranged
in parallel with each other. As a result, it was possible to align
and orient the cells in parallel. Further, in the case where the
cells in FIG. 7B were detached, the cells in each of the
island-like cell adhesivable portions formed one cell mass each,
and therefore a cell mass having a uniform size could be
obtained.
[0182] FIG. 8 is a photograph showing the results of optical
microscopic observation of the human iPS cells cultured in the
present Example. As a result, it was confirmed that the human iPS
cells can also be cultured in a patternwise manner. Further, in the
case where the cells in FIG. 8 were detached, the cells in each of
the island-like cell adhesivable portions formed one cell mass
each, and therefore a cell mass having a uniform size could be
obtained.
INDUSTRIAL APPLICABILITY
[0183] According to the present invention, it is possible to
provide a novel microstructure forming technique. More
specifically, the present invention is capable of providing a
method for producing a structure composed of crosslinked polymer, a
laminate for producing a structure composed of crosslinked polymer,
a structure composed of crosslinked polymer, a structure composed
of crosslinked polymer for cell culture, a cell culture method, a
method for producing a cell mass, and a cell mass.
REFERENCE SIGNS LIST
[0184] 10: substrate
[0185] 20: photo-dissoluble polymer layer having a photoacid
generating ability
[0186] 30: pregel composition
[0187] 31: crosslinked polymer sheet
[0188] 40: solvent (liquid)
* * * * *